Ribonucleotide reductases (RNRs), which provide the deoxynucleotide precursors for DNA synthesis by reducing the corresponding ribonucleotides, are divided into three classes on the basis of the cofactor that initiates catalysis. Historically, class I, which comprises the RNRs from aerobic bacteria such as Escherichia coli (Ec) and higher eukaryotes including humans, is further subdivided into three subclasses that are distinguished, in part, by the identity of the dimetal cofactor (Fe2, Mn2, and MnFe for subclasses a, b, and c, respectively) in the ? subunit that either directly or indirectly generates a consered cysteine radical (Cys?) initiator in the a subunit. It has been established for the archetypal clss Ia Ec RNR that Cys? generation occurs by a conformationally gated, long distance proton-coupled electron transfer (PCET) mechanism, and important details of this step have been defined. The research plan herein aims to elucidate the PCET mechanisms in the Ib and Ic RNRs at an equivalent level of detail. This goal will be achieved through trapping strategies that permit the PCET product state to be compared to the resting (reactant) state by Mssbauer, electron paramagnetic resonance, and magnetic circular dichroism spectroscopic techniques. In addition, our preliminary evidence suggests the existence of a previously unrecognized, fourth subclass (class Id), in which the cofactor's composition, structure, and mechanism of Cys? generation remain to be determined. As even a basic understanding of the class Id RNRs is lacking, multi-turnover kinetic assays will be optimized for this enzyme class, and used in conjunction with single-turnover experiments and spectroscopic analysis of cofactor composition to identify the functional cofactor. PCET reactant and product states of the active cofactor will b interrogated crystallographically and spectroscopically to define their structures and the mechanism of radical initiation. Here again, trapping strategies employing site-directed mutagenesis, chemical reductants, and/or mechanism-based inactivators will enable preparation of the PCET product state both to identify the functional cofactor and define its structure.